XYLO-A N D ARABINOSURANOSYLTHIOGUANINE of 6-chlorc-2-fluoropuririe (I, 1.5 g, 8.6 mmoles) in 25% methanolic Me3N was kept a t 25". After a few min a cryst ppt appeared. The mixt was kept a t 25' for 48 hr; the ppt was collected and washed with EtOH to yield colorless plates (0.45 g, 26%), mp 300°, of 2-fluoro-6-trimethylaminopurine betaine ( X X ) : uvmaX(pH 1.0) 270.5 ( e 7.9 X lo3),(pH 6 ) 273 (7.3 X IO3), (pH 12) 273 nm (7.6 X IO3). Anal. (CBH11NsF) C , H, N, F . The filtrate of the above reaction, after evapn to dryness under reduced pressure, gave a cryst residue consisting of 2-fluoro-6dimethylaminopurine (XIX), 1.1 g (70%), mp 220". Anal. (C7H8N5F)C, H, N, F. This material was identical with the product prepared by Montgomery and Hewson.6 Reaction of 2-Fluoro-6-chloropurine (I) with Hydrazine.-A 10% hydrazine hydrate ethanolic soln (25 ml) was added t o 2-fluoro-6-chloropurine (I, 1.5 g, 8.7 mmoles) dissolved in EtOH (25 ml) a t 5". After stirring a t '5 for 1 hr, the resulting ppt was collected by filtration, and dried to yield 2-fluoro-6-hydraeinopurine (XXI), 1.3 g (87'%) of thin needles, mp 142'. Anal. (C5HaNe.F) C, H, N, F. When X X I (20 mg) was boiled in HzO (5 ml) and Raney Xi (50 mg) for 2 hr, the resulting soln showed uv spectra and Rr values identical with those of 2-fluoroadenine (VIII). 6,6-Bis(2-fluoroadenine) (XXII).-Solns of 2-fluoro-6-chloropurine ( I , 1.20 g, 6.9 mmoles) in EtOH (25 ml) and 2-fl~oro-6hydrazinopurine ( X X I , 1.15 g, 6.9 mmoles) in 70y0 aq EtOH (2,j ml) were combined. Anhyd NaOAc (0.67 g, 7.6 mmoles) was added, and the mixt was refluxed for 6 hr and kept at 25' overnight. The ppt which formed was collected by filtration and repeatedly washed with H 2 0 and EtOH to yield 1.03 g (quant) of a yellow microcryst product, mp >300'. Anal. (C10HCNIOF2) C, H, K. F test was positive. Treatment of XXII with Raney Ni.-A suspensioii of XXII (0.5 g, 1.6 mmoles) in H20 (25 ml) and Raney Ni ( 3 g) was refluxed for 12 hr. The reaction mix was filtered when hot, the Ni was washed with boiling HzO, and the combined filtrates were evapd to dryness under reduced pressure. The residue was suspended in H2O ( 5 ml), filtered, and dried to yield 70 mg (147,) of 2-fluoroadenine (VIII). 2-Hydroxylamino-6-aminopurine ( 2-Hydroxylarninoadenine) (XXIII).-A suspension of VI11 (0.30 g, 1.8 mmoles) in 0.6 M ethanolic HONHZ(300 ml) and 0.3 ml of 30% aq soln of HOKH2. HClZ2was refluxed for 6 hr and kept a t 25" overnight. The (22) A . Giner-Sorolla, S. A. O'Bryant, J. H . Burchenal, and A. Bendich, Bzochemzstry, I , 3057 (1966).
Journal of Medicinal Chemistry, 1971, Vol. 14, No. 9 819 resulting ppt was collected by filtration, washed with HzO, and dried to yield 0.17 g (53%) of microneedles: mp 270' dec; uvmax(pH 3.0) 242 (shoulder) ( e 12.2 X lo3), 280 (10.1 x l O 3 ) , (pH 7.0) 253 (8.6 X lo3), 274 (9.1 X lo3); pKa = 4.74 ( f 0 . 0 4 ) . Anal. ( C ~ H ~ N ~ O . O . ~ C, ~ HH, ZO N .) XXXIII gave blue-green color with FeCls soln (HONH) and deep orange-brown color with 1 AT NaOH (azoxy formation). Boiling of XXIII with 5% ethanolic hydrazine for 3 hr gave a soln with uv spectra and Rf values identical with those of 2,bdiaminopurine (XVIII). 2-Hydroxylaminopurine (XXV).-A soln of 2-fluoropurine6 (XXIV, 1.2 g, 8.5 mmoles) in 1 M ethanolic HONHz was refluxed for 10 hr and kept a t 25" overnight. The resulting ppt was collected and washed with cold HzO t o yield a pale yellow cryst material, 0.83 g, mp 260' (exploded when inserted a t 250"). Upon concn of the filtrate t o about 30 ml, a second crop (0.31 g, mp 260°, expl) was obtd (yield, 87%). An anal. sample was obtd by thorough washing of the first ppt with 90% aq MeOH a t 23': uvmaX(pH 7.0) 233 (E 8.6 X lo3),346 nm (13.6 X 103); pK,, = 2.08 (f0.0f5), pK,, = 8.52 ( h O . 1 ) . Anal. (C5HaNsO. 0.33H20) C, H, N. A suspension of XXV gave an intense dark blue color with FeC4 soln. When XXV was dissolved in 2 Y KaOH an orange soln was obtd, but in contrast to &hydroxylaminopurine,2 no ppt of the corresponding azoxy derivative was observed. The uv spectra of this soln showed profound decompn. A sample of XXV (10 mg) was dissolved in 5 7 , aq NH3 ( 5 ml) and Raney Ni (50 mg) was added. After boiling for 15 min the resulting soln showed uv spectra and Rr values identical with those of 2-aminop~rine.'~
Acknowledgments.-The authors wish to acknowledge Dr. Harry B. Wood, Jr., Xational Institute of Health, Bethesda, Md., for a generous gift of thioguanosine and screening tests, and Dr. C. Chester Stock and G. Tarnonslii for chemotherapeutic work on Ridgeway osteogenic sarcoma. They also wish to express their gratitude to Dr. A . Bendich, Dr. J. J. Fox, and Dr. 11. L. Cox for stimulating discussions and t o Mrs. M, B. Rleyers, Rlrs. J. Longley-Cook, and Miss 11. McGravey for excellent technical assistance.
Xylo- and Arabinofuranosylthioguanine and Related Nucleosides Derived from 2-Acetamido-6-chloropurine' WILLIAM W. LEE,* ABELARDO P. ~ I A R T T N ROBERT EZ, W. BLACKFORD, VICTOR J. BARTUSKA, ELMER J. REIST,AND LEONGOODMAN' Life Sciences Research, Stanford Research Institute, Menlo Park, California 94026 Received February 1, 1971 9-(p-D-Xylo- and 9-(a- and p-D-arabinofuranosyl)thioguanine (1, or-5, and p-5) have been synthesized. The Hg derivative of 2-acetamido-6-chloropurine gave 9-(2,3,5-tri-0-benzoyl-or-~-arabinofuranosyl)and 9-(2,3,5tr~-0-acetyl-~-~-xylofuranosyl)-2-acetam~do-6-chloro-9H-pur~ne (9 and 10, respectively) on reaction with the appropriate halo sugar. Treatment of 9 and 10 with NaSH and deacylation gave 0-5 and 1, respectively. Compd 10 was converted to the xyloside of 2-amino-6-chloropurine (11) and guanine (2). Both of these could be converted through several intermediates to the 2',3'-anhydronucleoside intermediate 15g. Cleavage with NaOAc in aq DMF afforded 9-(4-D-arabinofuranosy1)guanine (p-6). Appropriate acylation, followed by thiation and deacylation, gave p-5, which was active against leukemia L1210 in mice; the other nucleosides tested were inactive.
Many compounds with antitumor activity have been found among purines and nucleosides. Thioguanine (1) This work was performed under the auspices of Chemotherapy, National Cancer Institute. National Institutes of Health, Public Health Service, Contract N o . PH-43-64-500. The opinlons expressed here are those of the authors and do not necessarily reflect those of Chemotherapy.
and thioguanosine, for example, are useful carcinostatic agents.2 Certain thioguanine nucleosides synthesized in these laboratories+-e.g., 3'-deoxythioguano(2) J. A. Stock in "Expenmental Chemotherapy," Vol. IV, R . J. Schnitzer and F . Hawking, E d . , Academic Press, New York. 1966, pp 134-35.
820 Journal of Medicinal Chemistry, 1971, Vol. 14, X o . Y
sine13,4a"J3'-O-methylthioguanosine,3~4a~c and both anomer8 of 2'-deoxythioguanosine4~~b~5-haveshown antitumor activity. Particularly interesting is a-2'-deoxythioguanosine, which is the first a-nucleoside n-ith antitumor activity. This cy-nucleoside is incorporated into the DNA of murine tumor cells.6 Other nucleosides of fraudulent sugars have shon n antitumor activity. Thus, the P-D-arabinofuranosides of c y t o ~ i n e ,6-mercaptopurine17a ~ and adeninega have demonstrated antiturnor activity, a b have the 0-Dxylofuranosides8b of adenine and 6-mercaptopuri~ie.~ These facts suggest that 9-(P-D-xylofuranosyl)thioguanine (1) and the a and /3 anomers of g-(D-arabinofuranosy1)thioguanine ( 5 ) be synthesized for antitumor evaluation. The synthesis and some bioassay results are reported here. Other biological data have been published already for 1;lO further studies of 2 and p-5 are in progress in other laboratories. X x
R
O
C '
W
RO
l , X =S;R = H 2, X = 0 R =H 3,X = S; R = Ac 4,X=OR=Ac
Cl S,R=Bz
"
O
c
a
RO 5, X = S R = H 6, X = O R = H 7,X = S; R = Ac 8, 0; R = A C
x'=
10,R = Ac 11, R = H
For the synthesis of 1 and a-5, use of the Hg derivative of 2-acetamido-6-chlor~purine~seemed an attractive routes3 Its application t>o the synthesis of a-5 was straightforward. Thus, the reaction of the Hg derivative of 2-acetamido-6-chloropurine and 2,3,5tri-0-benzoyl-D-arabinofuranosyl chloride" gave the (3) G . L. Tong, E;. J. Ryan, W. W.Lee, E. 11. .icton, and L. Goodman, J . Ore. Chem., 32, 859 (1967). (4) (a) These compds were screened for antitumor activity i n t h e mouse leukemia L1210 systems under the auspices of Chemotherapy, Xational Cancer Institute, according t o t h e protocol in Cancer Chemuther. R e p . , $6, 1 (1962); (b) A . Goldin, H. B. Wood, J r . , and R. R. Engle, i b i d . , S u p p l . , 1, 1 (1968); ( c ) unpublished result. (5) It. €€. Iwamoto, E. M . Acton, and L. Goodman, J . ' l f e d . C h e m . , 6, 684 (1963). (6) G. A . LePage, C a n . J . B i o c h e m . , 46, 655 (1968). (7) (8) A. P. Kimball, B. Bowman, P. S. Bush, J . Herrior, and G . A . LePage, Cancer Res., 26, 1337 (1966); (b) P. J . Burke, A . H. Owens, Jr., J. Colsky, B. I. Shnider, John H. Edmonson, A . Schilling, Harvey S. Brodovsky, H. J. Wallace, Jr., and T. C. Hall, ibid., 30, 1512 (1970). (8) (a) J. J. Brink and G. A . LePage, C a n . J . Biochem., 45, 1 (1965); (b) D. B. Ellis and G. -4.LePage, Mol. Pharmacal., 1, 231 (1965). (9) K. Sato, G . A. LePage, and A. P. Kimball, Cancer Res., 26, 741 (1966). (10) T. Kaneko and G . A . LePage, ibid., 3 0 , 699 (1970). (11) A. K. Bhsttaoharya, R . K. Ness, and H. G. Fletcher, J r . , J . O r g . C h e m . , 28, 428 (1963).
LEI:, f't a/.
blocked a-nucleoside 9, TI hicL was treated with 2mercaptoethanol and base3 t o afford g-(cy-D-arabinofuranosy1)guanine (cy-6), and TI ith S a S H in l I e O H t o give 9-( a-n-arabinofuranosy1)thioguanine (cy-5). The synthesis of S-(~-D-arabinofuranosyl)thioguanine (p-5) seemed feasible either b l (a) transformation of P-D-xylofuranosylthioguariiIie (1) vaa the epoxide 15t1?t o 6-5. or (b) by tliiation of P-arabinofuranosylguanine @-6)l3 that was suitably protected (e.c/., as P-8). The required amounts of p-6, riot attainable by the original synthesis,l 3 seemed available by the transformation 9f 9-(~-D-x?.lofura~iOS~1)gUariine (2) z za the epoxide 15g to 6. Either route (a or b) required that large amount5 of 10 be accessible. Reaction of the Hg derivative of 2-acetamidochloropurine n ith L',3.vY-tri-O-acetyl-n-xylofuranosj1 bromide14 afforded the blocked nucleoside 10, IT hich required purification by Florisil column chromatography. Sucleoside 10 n as converted 11 ith HS(CH,)2O H and baie to 9-(P-~-xylofuranosyl)guanine (2) ; I 4 v, ith KaSH and base to the X J lofuranosylthioguanine 1; or nitli methanolic S H 3 to 11. Large amounts of these interinediateq 13 ere available by this route. iittempts to convert 1 to P-5 proceeded satisfactorily through the initial steps. Because of good solubility in acetone, 1 could be smoothly acetonated to the isopropylidene derivative 12t (see Scheme I) using EtS03H rather than the less available di-p-nitrophenyl 11) drogen phosphate.lq llesylation of 12t resulted in the uptake of more than one mesyl group, but, after base treatment n i t h S a H S or in the presence of mercaptoethanol the meyyl derivative 13t TI as obtained. C173COrH converted 13t to 14t. ~ h i c h yielded the epoxide 15t on treatment with S a O J I e Efforts to convert 15t to P-5 uith PhCOJa in 93% aq D l I F gave a n unideritihed product that, although not a disulfide, had lost the properties of a thioguanine. An authentic disulfide (17) derived from xylofuranosylthioguanine (1) n as prepared for comparicon The desired 8-5 waq finally obtained by route b The aminochloropurine nucleoside 11 was readily acetonated (to 12c, Scheme I) and then mesylated to give 13c. Reaction n i t h HS(CHz)?OH and base afforded 13m, v,liich, nhen refluxed several hour5 in 70y0 HOAc, gave the mes) 1 guanine nucleoside 14g. Treatment with methanolic S a O l l e yielded the epoxide 15g, 11 hich n as readily isolated as the S a salt. Either SaOAc or S a 0 2 C P h in hot 9;yc aq D l l F opened the epoxide 15g at (2-3' to give p-6. S o C-3' opening (300 221-223 dec 210-2 12 195-196 97-112 h 282-284 (278-280) 243-246 dec (243-246 dec) 119-120 246-247(242-244) 204-205 229-231 (229-231) h 203-204
ChromatogC
Solvb
Compd
0.61T A 0.15 P B 0.60PE 0.90T A C.58 TA 0.35 P H 0.63PE 0.67TA
C W
E EA
0.80 T B 0.56 T D
C-DM F-S E- W W
0.73 TA 0.66T C 0.42 TA 0.77 TA
EA-S bI
0.84 T C 0.64 T A 0.57TA 0.60T C 0.54 TA 0.71 T A 0.26TA
E-W EA W
11
0.56PE 0 . 55 TA W 33 (52) >300 dec 0.45 TA W 93 207-208 M 0.57TA 60 Yield data and melting point values are for anal. samples except values in parentheses, which are for homogeneous products suitable for the next reaction. b Crystnor triturationsolvents are: D l I , 1,2-dimethoxyethane; C, CHC13, L), DLlF; E, EtOH; EA, EtOA4c; F, EtzO; SI, XIeOH; S, Skellysolve B (essentially hexane, bp 60-68");W, H20. Solvent systems for tlc are MeOH-EtOAc in various ratios: T A (30:70),T B (20:80),T C (10:90);TL) is CHC13-EtOAc (65:35). Solvent sybtems for paper chromatography are: PB, n-BuOH-HZO (saturated); PE,5 7 , NaZHPOa, p H 8.9; PH, n-BuOH-HOAc-HzO (5:2:3). Where analyhes are indicated only by symbol of the elements, anal. results obtained were within 4~0.4% of theoretical values. e Overall yield from 15g. f Overall yield from P-6. After column chromatography. Solid foam. As the N a salt.
1st 17 18
1
13g was more satisfactory with hot aq AcOH than with CF&02H. The P-arabinofuranosylguaninep-6 could be acetylated (to p-8), then heated in pyridine with PzSi t o afford 0-7, and deacylated with methanolic N a O l l e t o yield 0-5. 0-5 has been obtained in 45% yield from the N a salt of the epoxide 15g without isolating the intermediates. The route via 2 provided the best overall yield. I n the Experimental Section, only representative procedures are given. The properties of the compds and intermediates are summarized in Table I. The antitumor activity of these compds was determined against L1210 mouse l e ~ k e m i a . ~ "The results for the target nucleosides, summarized in Table 11, show that p-D-arabinofuranosylthioguanine (p-5) is active and has low toxicity. The other compds in Table I are not active against L1210; however, other biological activity has been reported for l , l o and 2 is under study by other laboratories. At doses of 400 mg/kg, the intermediates 3, CY-7 (200mg/kg), 9, 11, 12c, 12g, 12t, 13c, 13m, 13t, 14c, 14t, 17, and 18 were inactive in the L1210 test. Compound 4 was inactive against KB cells in tissue culture. Experimental Section Melting points were determined in a Fisher-Johns apparatus and were not corrected. Optical rotations were measured with a
Perkin-Elmer Model 141 automatic polarimeter. Paper chromatograms were run by the descending technique on Whatman No. 1 paper. Tlc Rere run on silica gel H F (E. Merck AG Darmstadt). The solvent systems are listed in Table I. All spots were detected by uv light. All solns were dried (MgSOa) and were concd in a rotatory spin evaporator zn vacuo with a bath temp of
